1. Technical Field of the Invention
The present invention relates to a method for preparing a reconstructed epidermis or a skin equivalent supplemented with at least one derivative of ceramide 7 and/or 5.5, comprising introducing at least one derivative of ceramide 7 and/or 5.5 into the culture medium for said reconstructed epidermis or said skin equivalent and/or in topically applying to said reconstructed epidermis or said skin equivalent a composition based on lipid lamellar vesicles incorporating at least one derivative of the ceramide 7 and/or 5.5 family.
The invention also relates to a reconstructed epidermis or a skin equivalent containing at least one derivative of the ceramide 5.5 family.
The present invention also relates to a composition comprising a dispersion, in an external aqueous phase, of vesicles formed by lipid lamellar phases separated from one another by hydrophilic layers, said lamellar phases comprising at least one amphiphilic lipid, and at least one derivative of the ceramide 7 and/or ceramide 5.5 family included in said lipid lamellar phases, and also to the use of said composition for reinforcing the barrier function of normal human epidermis, and improving the barrier function of an epidermis exhibiting even a slight deficiency in 6-hydroxy-4-sphingenine-base ceramides, in particular of dry skin and of reconstructed skin or skin equivalents.
2. Description of Background and/or Related and/or Prior Art
Human skin consists of two compartments, namely a deep compartment, the dermis, and a superficial compartment, the epidermis. The epidermis is in contact with the outside environment. Its role consists in protecting the organism against dehydration and outside attacks, whether they are chemical, mechanical, physical or infectious.
The natural human epidermis is composed mainly of three types of cells, which are keratinocytes, present in great majority, melanocytes and Langerhans cells. Each of these cell types contributes by virtue of its own functions to the essential role played in the organism by the skin.
The cells constituting the epidermis are delimited by a lipid domain. During differentiation, the phospholipids, the role of which consists in developing the fluid structure of the cell membranes of the living layers of the epidermis, are gradually replaced with a mixture composed mainly of fatty acids, cholesterol and sphingolipids.
These lipids are organized in specific lamellar liquid crystal phases, the integrity of which depends not only on the quality of the fractions present but also on their respective proportions. This lamellar structure of the lipids of the lipid domain of the epidermis is responsible for the epidermal barrier function.
Epidermal lipids are synthesized mainly in the living epidermis. They consist mainly of phospholipids, ceramides (or sphingolipids), cholesterol, free fatty acids, triglycerides, cholesterol esters and alkanes.
Ceramides are one of the essential constituents of epidermal lipids, making it possible, partly, to provide the lamellar liquid crystal structure thereof, but also the barrier function of the epidermis.
Ceramides are made up of a sphingoid base, which may be of four types, sphinganine, sphingenine, phytosphingosine and 6-hydroxy-4-sphingenine, and of a fatty acid which may be saturated, α-hydroxylated or ω-esterified. The various possible combinations between bases and fatty acids result in about ten ceramides listed by Robson, K. J.; Stewart, M. E.; Michelsen, S.; Lazo, N. D.; Downing, D. T., “6-hydroxy-4-sphingenine in human epidermal ceramides,” in J. Lipid Res., 1994, 35:2060–2068; and Chopart M., Castiel-Higounenc I., Arbey E., Guey C., Gaetani Q., Schmidt R., “The Normal Human stratum corneum: a new ceramide profile,” Perspectives in Percutaneous Penetration, 8th International Conference, Antibes Juan-Les-Pins—France, Apr. 2–6, 2002.
It has been possible to develop models which are more or less close to human skin. Mention may, for example, be made of the models described in EP-A-285471, EP-A-285474, EP-A-789074, EP-A-502172, EP-A-418035, WO-A-9116010, EP-A-197090, EP-A-20753, FR-A-2665175, FR-A-2689904 and FR-A-2792650.
Very generally, the models of reconstructed skin described in those documents comprise human keratinocytes possibly combined with other skin cells such as melanocytes and/or Langerhans cells, deposited on a support, often a dermis equivalent, and cultured under conditions such that they enter into a program of differentiation resulting in the formation of an epidermis equivalent. The dermis equivalents described to date are either artificial membranes, such as, for example, filters of the Millipore trademark, subcutaneous substitutes based on collagen, plastic or any other support compatible with cell viability, or supports which are more developed in order to make them closer to natural dermis, such as pre-de-epidermalized dermis or collagen/fibroblast mixed lattices. In the collagen/fibroblast mixed lattices, the combination of native collagen and isolated human fibroblasts leads to a dermis equivalent being obtained which mimics a dermis which has not been subjected to the action of time.
Reconstructed skin models generally exhibit a deficient barrier function (M. Ponec, P. J. J. Wauben-Penris, A. Burger, J. Kempenarr, H. E. Bodde, Skin Pharmacol., 1990; 3: pp. 126–135). This deficiency is largely due to important modifications in the ceramide profile of this model which have been observed compared to a normal human epidermis.
In addition, it is known from the prior art that atopic, xerotic and aged skin may be associated with a decrease in synthesis of ceramides 1 and/or 3. Mention may, for example, be made of the following documents:
Imokawa et al., in J. Invest. Dermatol., 96 (4): 523–6, 1991, disclose that ceramides are involved in the barrier function and that their synthesis, in particular that of ceramide 1, is decreased in the case of atopic and xerotic skin.
Di Nardo et al., in Acta Derm. Venereol., 78(1): 27–30, 1998), describe atopic dermatitis as readily irritable and dry skin in which the barrier function is impaired. The authors showed that a decrease in the synthesis of ceramides 1 and 3 may be the cause of the dry skin and of the barrier function impairment in atopic dermatitis.
Rogers et al., in Arch. Dermatol. Res., 288:765–770, 1996, describe a decrease in the synthesis of ceramides, in particular ceramide 1, which contributes, in aged skin, to a disturbance of the barrier function and to xerosis, particularly during the winter months.
Rogers et al., in J. Invest. Dermatol., 100:510, 1993 disclose that the lipid multilayer structure of dry skin is disturbed, and that this disturbance is accompanied by an increase in free fatty acids and by a decrease in ceramides.
In order to improve aged skin, dry skin and skin sensitivity, compositions are already known which contain a precursor for ceramide synthesis selected from: the sphinganine and sphingosine bases, fatty acid amides, and vitamin B3 as a ceramide synthesis stimulator (WO 99/47114), as are compositions which contain an intermediate of the synthetic pathways, or precursor, for ceramides chosen from fatty acids, the sphinganine and sphingosine bases, and vitamin A as a ceramide synthesis stimulator (WO 94/23694).
M. Ponec et al., in Skin Pharmacol., 1990; 3: pp. 126–135, disclose, moreover, that reconstructed skin models generally exhibit a deficient barrier function. This deficiency is largely due to important modifications of the ceramide profile of this model which have been observed compared to a normal human epidermis.
It is also known practice, from FR-2,811,556, to use 6-hydroxy-4-sphingenine to enhance the barrier function of reconstructed skin and to reinforce the lipid barrier of the epidermis, in particular of dry and/or rough and/or damaged skin, and/or to re-establish or maintain the integrity of the stratum corneum, and/or to improve the surface appearance and/or moisturization of the skin, and/or to protect the skin, in particular dry and rough skin, and/or as a nutritive agent essential for keratin substances (skin, hair, eyelashes, nails), and/or to reinforce the lipid barrier of reconstructed skin or skin equivalent, and/or to enhance and/or maintain the lipid content of human epidermis, in vivo and in vitro, and/or to improve the quality and the properties, such as the lipid content and/or barrier property content of reconstructed epidermis and/or epidermal cell cultures.
However, in using 6-hydroxy-4-sphingenine, it is not possible to control the nature of the ceramide which will be obtained in situ subsequent to its combination with a fatty acid in the epidermis. 6-Hydroxy-4-sphingenine is in fact the constitutive sphingoid base of several ceramides: ceramides STAR, 4, 5.5 and 7 (“The Normal Human stratum corneum: a new ceramide profile” M. Chopart et al., Prospectives in Percutaneous Penetration, 8th International Conference Antibes Juan-Les Pins, France, 2–6 Apr. 2002 and document JP2000/143,598 by Kanebo). In addition, reconstructed skin is a fragile structure whose survival can only be maintained for a limited period of time (approximately 1 month). Any gain in time which allows reconstructed skin to acquire a barrier function is therefore an important parameter in relation to its lifespan.
In order to improve the lipid profile of reconstructed epidermis, it is also known practice to add ascorbic acid or vitamin C (J. Invest. Dermatol., 109:348–355, 1997) or ascorbic acid derivatives (FR-2,807,320) to the culture medium.
However, because of its chemical structure (alpha-ketolactone), ascorbic acid is very sensitive to certain environmental parameters such as light, heat and aqueous media, in particular alkaline and/or aerobic media. Because of these problems of stability, it is necessary to use high concentrations of ascorbic acid in order to observe the effect on the skin of a composition containing it. In addition, in introducing ascorbic acid or one of its derivatives into the culture medium, it is not possible to control the exact nature of the ceramide which will be synthesized in situ.
There remains therefore a need for other methods for obtaining more rapidly reconstructed skin models for which the ceramide profile is improved, with a barrier function which comes close to normal human epidermis.